This method can help answer key questions on protein localization and the dynamics in photosynthetic organisms. By combining the photobleaching method with super-resolution microscope, we are able to detect the protein dynamics of photosynthetic organisms in a remarkable detail. This method not only provides insight into the protein dynamics of Prochlorococcus.
It can also be applied to many other organisms containing radio-re-dop-sing and bacteriochlorophyll. Demonstrating this procedure will be Yanxin Liu, a graduate student from my laboratory. To begin, add five milliliters of seawater-based Pro99 medium to a culture flask and inoculate it with one milliliter of Prochlorococcus MED4.
Grow the Prochlorococcus at 23 degrees Celsius under a light with an intensity of 35 micromole photons per square meter. After five days, collect one milliliter of the culture into a 1.5 milliliter tube. Add 100 microliters of freshly prepared formaldehyde to the tube to fix the culture.
Incubate in the dark at room temperature for 20 minutes. Then, spin down the sample at 13, 500 times G for one minute. Remove the supernatant and resuspend the cells in 100 microliters of Pro99 medium.
Store the sample at four degrees Celsius in the dark until ready to perform immunostaining. First, vortex the vial of polystyrene beads. Using 50%ethanol, prepare a one-to-20, 000 dilution as a working slurry.
Turn on the hotplate and set the temperature to 120 degrees Celsius. Next, place the coverslips onto the hot plate. Load 100 microliters of the working slurry onto each coverslip and incubate the coverslips on the hotplate for 10 minutes.
Then carefully transfer the coverslips to Petri dishes bead-side up for storage at room temperature. When ready to coat the coverslips, retrieve one, making sure that it is bead-side up. Add 100 microliters of a one milligram per milliliter poly-L-lysine solution to the center of the coverslip and let it sit at room temperature for 30 minutes.
After this, use a pipette to carefully aspirate any unattached poly-L-lysine and transfer it to a 1.5 milliliter tube. Store this poly-L-lysine at four degrees Celsius for later use. Rinse the coverslip with 10 milliliters of ultra filtered water in a 60-millimeter Petri dish and then briefly dry it with a paper towel.
Transfer the coated coverslip to a new Petri dish with Parafilm at the bottom, which will be used as the staining dish. Load 100 microliters of the fixed sample onto the coverslip and let it sit for 30 minutes to allow cell attachment. Next, use a paper towel to absorb the remaining solution and then transfer the coverslip to a well in a 12-well plate for washing.
Add one milliliter of PBS buffer to the well to wash the coverslip. Then remove the PBS and add one milliliter of fresh PBS to wash the coverslip a second time. Use a pipette to remove the PBS and replace it with one milliliter of freshly prepared permeabilization buffer.
Incubate the washing dish at 37 degrees Celsius for 20 minutes. Then remove the permeabilization buffer. Begin the washing process by adding one milliliter of fresh PBS buffer.
Place the washing dish on a shaker to gently agitate the dish for five minutes. After this, remove the PBS and repeat the washing process three times. Transfer the washed coverslip to the staining dish.
Add 50 microliters of blocking buffer on the top of the coverslip. Place the entire staining dish on ice and then move it under the xenon light source for photobleaching for at least 60 minutes. After photobleaching and blocking, use the edge of a paper towel to remove the blocking buffer.
First, dilute one microliter of anti-FtsZ antibody into 99 microliters of blocking buffer. Place 50 microliters of the diluted primary antibody onto the coverslip and incubate it at room temperature for 30 minutes. Transfer the coverslip to a well of the washing dish.
To begin the wash, add one milliliter of PBS. Then transfer the washing dish to a shaker and gently shake it for five minutes. Remove the PBS and repeat the entire washing process three times.
Next, transfer the coverslip to the staining dish. Place 50 microliters of the diluted secondary antibody onto the coverslip and incubate it in the dark and at room temperature for 30 minutes. Transfer the coverslip back to the washing dish.
To begin the wash, add one milliliter of PBS. Wrap the washing dish in aluminum foil to protect it from light. Transfer the plate to the shaker and gently shake it for five minutes.
Remove the PBS and repeat the entire washing process three times. Immediately before the STORM imaging, prepare one milliliter of imaging buffer as outlined in the text protocol. Load the coverslip into the loading chamber.
Add the freshly prepared imaging buffer, being gentle to avoid washing off the cyanobacterial cells. After this, place a rectangular coverslip on top of the imaging buffer to prevent it from reacting with the oxygen in the air. Turn on the camera, the LED light, and the laser, and open the STORM software.
Add half a drop of immersion oil on top of the lens. Load the chamber, making sure that the objective lens is making contact with the coverslip. And examine the signal with a 750-nanometer laser.
Identify a sample area that contains both cells and fiducial markers. Then start the software for sample drifting correction. Acquire one wide-filed images as a reference, with the camera electron multiplication gain at 300 and an exposure time of 30 milliseconds.
Increase the 750-nanometer excitation laser intensity to approximately 4.5 kilowatts per square centimeter. Once the fluorophores have transitioned into a sparse blinking pattern, acquire one super-resolution image by collecting 10, 000 frames at 33 hertz. Then reconstruct the super-resolution images from the raw data as outlined in the text protocol.
In this study, STORM is used to achieve super-resolution imaging by activating individual photo-switchable fluorophores stochastically. The location of every fluorophore is recorded, and a super-resolution image is then constructed based on these locations. While the absorption spectra of Prochlorococcus peaks at 447 and 680 nanometers and has minimum absorption above 700 nanometers, Prochlorococcus MED4 cells still emit high autofluorescence when exposed to an extremely high intensity of the 750-nanometer laser, which is required for STORM imaging.
After using the photobleaching method described here for 30 minutes, the cells'autofluorescence is seen to decrease, although several cells with autofluorescence are still detected. Elongating the photobleaching to 60 minutes results in the majority of the cells losing their autofluorescence. These results indicate that the developed photobleaching method is capable of greatly reducing the autofluorescence of photosynthetic organisms.
After photobleaching, STORM is used to visualize the cell division protein, FtsZ, in the cells. Using STORM, a detailed morphology of the FtsZ ring is revealed. By rotating the 3D STORM images, four different types of morphologies were identified, clusters, an incomplete ring, a complete ring, and double rings.
It is important to remember that the light intensity and duration of the photobleach may differ for different organisms. After its development, this method paves the way for researchers who want to study protein organization and potential function in photosynthetic cells in detail.
